Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Amiloride sensitizes prostate cancer cells to the reversible tyrosine kinase inhibitor lapatinib by modulating Erbb3 subcellular localization

doi: 10.1007/s00018-024-05540-5

Figure Lengend Snippet: Amiloride promoted ErbB3 translocation from the nucleus to the cytoplasm and the plasma membrane in HSPC cells ( A ) Hormone-sensitive LNCaP cells were treated with varying concentrations of amiloride for 72 h before being lysed, fractionated and analyzed by immunoblot. ( B ) Immunofluorescence microscopy (IF) in LNCaP cells treated with DMSO or 75 µM or and probed with IF specific C-terminal ErbB3 antibodies or ( C ) 10µM or 25µM amiloride and probed with IF specific N-terminal ErbB3 antibodies for 72 h (scale bars = 30 μm). Note that vehicle treated LNCaP cells expressed nuclear ErbB3 (red) whereas amiloride-treated cells had significantly decreased ErbB3 expression in the nucleus (hollowed out). Location of nuclei are identified by blue DAPI staining. Plasma membrane localization of ErbB3 at cell-cell junction was also noted in amiloride-treated but not in vehicle treated cells. Note that both N- and C-terminal ErbB3 antibodies demonstrate lighter nuclear staining in amiloride-treated cells. ( D ) Cells were subjected to viability assays using the stated concentrations of amiloride. p-values are calculated with respect to DMSO

Article Snippet: After washing thrice with cold PBST, coverslips were inverted and mounted onto uncharged glass slides with UltraCruz Hardset Mounting Medium plus DAPI (SantaCruz BioTech, Dallas, TX).

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Western Blot, Immunofluorescence, Microscopy, Expressing, Staining

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Specific primers used for quantitative PCR.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques:

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Primary and secondary antibodies used for Western Blotting.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Western Blot, Membrane, Blocking Assay, Plasmid Preparation

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Physiological glutathione synthesis in liver of p40 wobbler mice. ( a ) mRNA expression levels of Glutaminase 2 ( Gls2) , Gclc , Gclm , and Gss from the distinct clinical phase (p40) of wild-type (WT) and wobbler (WR) liver were investigated by qPCR. Significantly increased mRNA levels of Gls2 , Gclc, and Gclm were observed in the liver of WR, while mRNA expression levels of Gss were not significantly altered. For relative quantification, the 2 −∆∆Ct method was performed using GAPDH for normalization. FC = Fold Change; N = 4. ( b ) Left: Semiquantitative analysis of protein expression levels in the liver of p40 WT and WR mice. GSS protein expression is increased in liver of WR, while protein expression of GCLC and GGT1 remains unchanged. Right: Representative Western Blots of GCLC (73 kDa), GSS (52 kDa), and GGT1 (55 kDa) in the liver of p40 WT and WR mice. Actin (42 kDa) and Calnexin (97 kDa) were used as loading controls, respectively. Bar charts represent the semiquantitative analysis of protein expression levels; N = 6. ( c , d ) Levels of tGSH ( c ) as well as the ratio of GSH/GSSG ( d ) in liver of p40 WR mice show no significant alterations compared to WT. tGSH, GSH, and GSSG levels of p40 WR mice compared to WT mice were analyzed via a fluorometric glutathione assay; N = 4. ( e ) ROS levels in liver tissue of p40 WR mice are unchanged compared to age-matched WT controls. ROS levels in liver tissue of p40 WR and WT mice were analyzed via a fluorometric assay; N = 4. All data are provided as mean ± SEM. Data were tested for significance using Student‘s t -test. Significant differences are indicated by ns. p ≥ 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Expressing, Quantitative Proteomics, Western Blot, Glutathione Assay

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Altered glutathione metabolism and synthesis in the cervical spinal cord of wobbler mice. ( a ) mRNA expression levels of Mrp4 , Mrp1 , Eaat3 , Slc7a11 , Slc3a2, and Slc25a11 in the cervical spinal cord of WT and WR mice from the distinct clinical stage (p40) were investigated by qPCR. mRNA levels of Mrp1 and Slc7a11 are significantly increased, while mRNA expression levels of Eaat3 are significantly decreased. Unaltered mRNA levels of Mrp4 , Slc3a2 , and Slc25a11 ; N = 4–8. ( b ) Upregulation of genes involved in glutathione cleavage in the cervical spinal cord of wobbler mice (p40). Significantly increased mRNA levels of Anpep and Ggt6 were measured, while mRNA expression levels of Ggt1 and Ggt7 are unaltered; N = 4. ( c ) Decreased mRNA expression levels of Gclc and Gss , alongside unaltered mRNA expression levels of Gls1 and Gclm in the cervical spinal cord of WR mice compared to WT mice age p40. For relative quantification, the 2 −∆∆Ct method was performed using GAPDH for normalization. FC = Fold Change; N = 4. ( d ) Left: Semiquantitative analysis of protein expression levels in the spinal cord of p40 WT and WR mice. Right: Representative Western Blots of GCLC (73 kDa),GSS (52 kDa), and GGT1 (55 kDa) in the cervical spinal cord of p40 WT and WR mice. Actin (42 kDa) and Calnexin (97 kDa) were used as loading controls, respectively; N = 4–8. All data are provided as mean ± SEM. Data were tested for significance using Student‘s t -test. Significant differences are indicated by ns. p ≥ 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Expressing, Quantitative Proteomics, Western Blot

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Physiological glutathione metabolism in hippocampus of wobbler mice. ( a ) mRNA expression levels of GSH transport relevant genes, Mrp4 , Mrp1 , Slc7a11 , Eaat3 , GSH cleavage relevant genes, Ggt1 , Ggt6 , Anpep , and GSH synthesis relevant genes, Gclc , Gclm , Gss , in the hippocampus of WT and WR mice from the distinct clinical stage (p40) were investigated by qPCR. Significantly increased mRNA levels of GSH transport-relevant genes were measured, while mRNA expression levels of Ggt6 were significantly decreased. For relative quantification, the 2 −∆∆Ct method was performed using GAPDH for normalization; N = 4. ( b ) Left: Semiquantitative analysis of protein expression levels in the hippocampus of p40 WT and WR mice. Right: Representative Western Blots of MRP4 (159 kDa), GCLC (73 kDa), GSS (52 kDa), and GGT1 (55 kDa) in the hippocampus of p40 WT and WR mice. Actin (42 kDa) and Calnexin (97 kDa) were used as loading controls; N = 4. ( c , d ) tGSH ( c ) as well as [GSH/GSSG]-ratio ( d ) in hippocampus of p40 WR mice compared to WT mice was analyzed via a fluorometric glutathione assay. Neither tGSH nor the ratio of GSH/GSSG in hippocampus of WR mice is significantly altered compared to WT mice, age p40. N = 4. ( e ) ROS levels in hippocampus of WR and WT mice were detected via a fluorometric assay at the developmental stage p40. WR hippocampus shows significantly elevated levels of ROS compared to WT; N = 4. All data are provided as mean ± SEM. Data were tested for significance using Student‘s t -test. Significant differences are indicated by ns. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Expressing, Quantitative Proteomics, Western Blot, Glutathione Assay

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Physiological glutathione metabolism in cerebellum of WR mice. ( a ) mRNA expression levels of GSH transport relevant genes, Mrp4 , Mrp1 , Slc7a11 , Eaat3 , GSH cleavage relevant genes, Ggt1 , Ggt6 , Anpep , and GSH synthesis relevant genes, Gclc , Gclm , Gss , in the cerebellum of WT and WR mice from the distinct clinical stage (p40), were investigated by qPCR. Significantly increased mRNA levels of GSH transport, some cleavage relevant genes, as well as Gclc but no other GSH synthesis relevant genes. For relative quantification, the 2 −∆∆Ct method was performed using GAPDH for normalization; N = 4. ( b ) Left: Semiquantitative analysis of protein expression levels in the cerebellum of p40 WT and WR mice. Right: Representative Western Blots of MRP4 (159 kDa), GCLC (73 kDa), GSS (52 kDa), and GGT1 (55 kDa) in the cerebellum of p40 WT and WR mice. Actin (42 kDa) and Calnexin (97 kDa) were used as loading controls; N = 4. ( c , d ) tGSH ( c ) as well as [GSH/GSSG]-ratio ( d ) in cerebellum of p40 WR mice compared to WT mice was analyzed via a fluorometric glutathione assay. Neither tGSH nor the ratio of GSH/GSSG in cerebellum of WR mice is significantly altered compared to WT mice at the age of p40; N = 4. ( e ) Unaltered levels of reactive oxygen species in cerebellum of wobbler mice compared to wild-type were detected via a fluorometric assay at the developmental stage p40; N = 4. All data are provided as mean ± SEM. Data were tested for significance using Student‘s t -test. Significant differences are indicated by ns. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Expressing, Quantitative Proteomics, Western Blot, Glutathione Assay

Journal: Antioxidants

Article Title: Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/antiox12051014

Figure Lengend Snippet: Proposed mechanism of dysregulated glutathione metabolism and synthesis in cervical spinal cord of wobbler mice. Glutathione metabolism between astrocytes and neurons: astrocytes import glutathione (GSH) via MRP4 (Multidrug Resistance Protein 4), while neurons do not contain an uptake mechanism for GSH and therefore rely on their own GSH synthesis. Both astrocytes and neurons can synthesize GSH from its constituent amino acids. Astrocytes can import cystine via the cystine/glutamate exchanger system Xc-. Astrocytes supply neurons with substrates for GSH synthesis. They export GSH via MRP1 (Multidrug Resistance Protein 1). In the next step, the ectoenzyme GGT (Gamma-Glutamyl Transferase) hydrolyzes GSH. The resulting Cysteinyl–Glycine (Cys-Gly) is cleaved by the ectoenzyme Aminopeptidase N (ANPEP). Next, the resulting amino acids are imported into neurons by different transporters, where they can be used for GSH synthesis. The main transporter for cysteine uptake in neurons is EAAT3 (Excitatory Amino Acid Transporter 3). Proposed mechanism of dysregulated glutathione metabolism and synthesis in the cervical spinal cord: a decreased protein expression of GCL (γ-Glutamyl-cysteinyl-ligase) leads to a decreased synthesis of γ-Glu-Cys (γ-Glutamyl-Cystein), resulting in a decreased GSH synthesis, even though protein expression of GSS (glutathione synthetase) is increased. Thus, less GSH is available for export from astrocytes via MRP1 and cleavage by GGT and ANPEP. As a compensatory mechanism, GGT1 is upregulated. However, EAAT3 is downregulated, which aggravates the deficient cysteine supply to neurons. Overall, this leads to an insufficient GSH synthesis in neurons. Due to the inadequate GSH levels, neurons’ and astrocytes’ defense against ROS (reactive oxygen species) is severely impaired. In astrocytes, increased levels of ROS lead to an increased expression of Slc7a11 encoding for the light chain of system Xc-, enhancing cystine import in astrocytes. Legend: Green arrows indicate an upregulation; red arrows indicate a downregulation of corresponding enzyme on protein or mRNA levels. Cys-Cys: Cystine; Glu: Glutamate; Gln: Glutamine.

Article Snippet: Anti-GGT1 mouse monoclonal IgG 2b antibody , #sc-374495, Santa Cruz, USA , 1:1000 in 5% BSA (#8076.2, Roth) in TBS-T , Nitrocellulose membrane (#1704270, biorad).

Techniques: Expressing